1. Field of the Invention
The present invention relates to a long-chain chondroitin sugar chain, a method for producing the long-chain chondroitin sugar chain, and a method of promoting chondroitin synthesis.
2. Description of the Related Art
First, abbreviations used in the present application are described.
CH:chondroitinCS:chondroitin sulfateHA:hyaluronic acidGlc:glucoseGluUA:glucuronic acidGlcNAc:N-acetyl glucosamineGalNAc:N-acetyl galactosamineGPC:gel permeation chromatographyHPLC:high performance liquid chromatographyK4CP:chondroitin polymerase derived fromEscherichia coli K4 strainMALDI-TOF-MS:Matrix Assisted Laser Desorption/Ionizationtime-of-flight mass spectrometryUDP:uridine 5′-diphosphate
CH is a kind of glycosaminoglycan in which GlcUA and GalNac are linearly and alternately bound by a β1-3 linkage and a β1-4 linkage, respectively. CH is present as CS proteoglycan in a cartilage and many connective tissues in an animal body, and plays important roles in cell adherence, cell generation, cell differentiation, nerve cell extension, cartilage formation, bone formation, tissue regeneration, and the like.
CS is commercially available as useful substances in the form of pharmaceuticals such as a tissue adherence prevention drug, an arthritis therapeutic drug, a drug for low back pain and arthragia, a neuralgia improvement drug, an omarthritis therapeutic drug, an eye dropper, a chronic nephritis therapeutic drug, and an analeptic, a health food, a cosmetic product (a moisturizer), and the like. In general, CS naturally exists as a sugar chain having a weight average molecular weight of 20,000 to 50,000, and it is known that CS's having a weight average molecular weight of 100,000 or more also exist. It is also known that those CS's have a structural characteristic such as a moisture rich characteristic and an ion retention characteristic, and have specific physiological function such as cell adherence and signaling of development and differentiation as an extracellular matrix component owing to their long-chain structure.
A plurality of CH synthetases derived from animals have been cloned. However, those expressed enzymes alone do not have a CH polymerase activity, or have a weak enzymatic activity, even if the enzymes have the CH polymerase activity, so the use of the enzymes does not sufficiently contribute to the efficient industrial production of CH sugar chains. On the other hand, K4CP has also been cloned, and it is known that the enzyme alone has a CH polymerase activity to produce CH efficiently (Patent Document 1 and Non-patent Document 1). However, CH synthesis reaction for a long period by using a recombinant purified enzyme of K4CP results in the production of CH sugar chains having only about 20,000 sugar chains.
Moreover, CH can also be produced by desulfation of CS. However, even if a sugar chain length of a starting material CS is long, sugar chains are cut by a side reaction. The present situation is that commercially available CH's have a weight average molecular weight of 10,000 or less.
A technology of synthesizing a long-chain CH sugar chain has not been known so far. However, from the view point of industrial usability, development of the long-chain CH sugar chain and a production method thereof are expected.    Patent Document 1: JP 2003-199583 A    Non-patent Document 1: Ninomiya, T. et al., 2002, Journal of Biological Chemistry, Volume 277, No. 24, p. 21567-21575